2′-岩藻糖基乳糖缓解LPS诱导的细胞炎症应答和屏障损伤的功能研究

Function of 2′-fucosyllactose in alleviating LPS-induced cellular inflammatory response and barrier damage

[目的]本文旨在探究2′-岩藻糖基乳糖(2′-FL)对脂多糖(LPS)诱导的仔猪小肠上皮J2细胞系(IPEC-J2)炎症和屏障功能的影响。[方法]以IPEC-J2为细胞模型,先通过预试验对LPS和2′-FL的处理浓度分别进行筛选,最终选择100μg·mL^(-1)LPS诱导细胞促炎反应,然后通过不同浓度(20和100μg·mL^(-1))2′-FL干预来探究其对LPS诱导IPEC-J2的促炎反应和屏障功能受损的调节作用。试验分为6组:对照组(CON)、LPS组(100μg·mL^(-1)LPS)、FL20组(20μg·mL^(-1)2′-FL)、FL100组(100μg·mL^(-1)2′-FL)、SF20组(100μg·mL^(-1)LPS+20μg·mL^(-1)2′-FL)和SF100组(100μg·mL^(-1)LPS+100μg·mL^(-1)2′-FL)。试验处理24 h后测定各组细胞活力、紧密连接蛋白、炎症因子和炎症相关通路基因的表达水平,通过Western blot检测NF-κB、Myd88、Claudin-1和Occludin蛋白的相对表达水平。[结果]与CON组相比,LPS组显著降低IPEC-J2细胞活力(P<0.05),显著上调促炎因子IL-6、IL-8及炎症通路NF-κB、Myd88和CD14基因的表达(P<0.05),显著下调抗炎因子IL-4基因的表达(P<0.05),显著下调紧密连接蛋白Claudin-1、Claudin-3、Claudin-4、ZO-1、ZO-2和Occludin基因的表达(P<0.05)。与CON组相比,FL20组还显著提高Claudin-1、Claudin-3、Claudin-4和ZO-2基因的表达水平(P<0.05)。与LPS组相比,SF20组和SF100组显著提高IPEC-J2细胞活力(P<0.05),显著下调IL-8及上调IL-4和IL-10基因的表达(P<0.05),显著下调NF-κB、Myd88和CD14基因的表达(P<0.05),显著上调Claudin-1、Claudin-3、Claudin-4、ZO-1、ZO-2、Occludin基因的表达(P<0.05)。Western blot结果显示,与CON组相比,只有LPS组的NF-κB蛋白显著上调(P<0.05)。与LPS组相比,SF20组的NF-κB、Myd88蛋白表达量降低,Claudin-1和Occludin蛋白表达量升高,但差异均不显著。[结论]LPS处理促进IPEC-J2细胞的促炎反应并造成屏障功能受损,2′-FL干预减弱了LPS刺激的促炎信号通路,改善了LPS引起的屏障功能受损。

[Objectives]This study was conducted to explore the effects of 2′-fucosyllactose(2′-FL)on lipopolysaccharide(LPS)-induced inflammation and barrier function in intestinal porcine epithelial cell line-J2(IPEC-J2).[Methods]Using IPEC-J2 as the cell model,the treatment concentrations of LPS and 2′-FL were screened respectively through pre-test,and finally 100μg·mL^(-1)LPS was selected to induce the cell pro-inflammatory response,and then the cells were interfered with by different concentrations(20 and 100μg·mL^(-1))of 2′-FL to explore its regulatory effect on LPS-induced pro-inflammatory response and the impaired barrier function of IPEC-J2.Therefore,the experiment was divided into six groups:the control group(CON),LPS group(100μg·mL^(-1)LPS),FL20 group(20μg·mL^(-1)2′-FL),FL100 group(100μg·mL^(-1)2′-FL),SF20 group(100μg·mL^(-1)LPS+20μg·mL^(-1)2′-FL)and SF100 group(100μg·mL^(-1)LPS+100μg·mL^(-1)2′-FL).After these groups were treated for 24 h,cell viability,gene expression levels of tight junction protein,inflammatory factors and inflammatory pathways in each group were determined,and the relative expression levels of NF-κB,Myd88,Claudin-1 and Occludin protein were detected by Western blot.[Results]Compared with the control group,LPS group significantly decreased the viability of IPEC-J2 cells(P<0.05);the expression of proinflammatory cytokines IL-6,IL-8 and inflammatory pathway NF-κB,Myd88,CD14 genes were significantly up-regulated(P<0.05);the gene expression of anti-inflammatory factor IL-4 was down-regulated(P<0.05);meanwhile,gene expression of tight junction proteins Claudin-1,Claudin-3,Claudin-4,ZO-1,ZO-2 and Occludin genes were significantly down-regulated(P<0.05).Compared with the control group,FL20 group also significantly increased the expression level of Claudin-1,Claudin-3,Claudin-4 and ZO-2 tight junction protein genes(P<0.05).Compared with LPS group,the viability of IPEC-J2 cells in SF20 and SF100 groups significantly increased(P<0.05);proinflammatory factor IL-8 was significantly down-regulated and anti-inflammatory factor IL-4 and IL-10 gene expressions were significantly up-regulated(P<0.05);inflammatory pathway genes NF-κB,Myd88 and CD14 were significantly down-regulated(P<0.05);tight junction protein genes Claudin-1,Claudin-3,Claudin-4,ZO-1,ZO-2 and Occludin were significantly up-regulated(P<0.05).Western blot results showed that compared with the control group,NF-κB in LPS group was significantly up-regulated(P<0.05).Compared with LPS group,the expression level of NF-κB and Myd88 proteins in SF20 group decreased,and the expression level of Claudin-1 and Occludin proteins increased,but the differences were not statistically significant.[Conclusions]LPS treatment promoted the pro-inflammatory response of IPEC-J2 cells and resulted in impaired barrier function,and 2′-FL intervention weakened the LPS-stimulated pro-inflammatory signaling pathway,thereby improving the LPS-induced barrier function impairment.